Transcript Viruses

Viruses
Microbiology 221
Viruses
 Obligate intracellular parasites
 They are able to reproduce their life cycle
only within the cell of their host
 They usually have an external capsid
composed of proteins
 Inner core of nucleic acid( dsDNA,
ssDNA,dsRNA, and ssRNA)
 Specificity for the host
Classification of viruses
 According to Baltimore classification, viruses are
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divided into the following seven classes:
dsDNA viruses
ssDNA viruses
dsRNA viruses
(+)-sense ssRNA viruses
(-)-sense ssRNA viruses
RNA reverse transcribing viruses
DNA reverse transcribing viruses
where "ds" represents "double strand" and "ss"
denotes "single strand".
Table of Viruses
Viral Capsids
Capsids
 Capsids are made from protein subunits
called capsomeres
 In some viruses, the capsomeres are all the
same Geometric shape in others the sub
units vary
Antigenic spikes
 Some viruses have molecules inserted into
the outer covering of the virus
 These may be glycoproteins
 In some cases these serve as a means of
attaching to the host cell
 They are specific for one cell type
Capsid Shapes
Ebola – Shepherd’s Crook
Enveloped Virus
Envelopes
 Lipid composition
 Acquired from the host when the virus
exits the cell
 Provides a means for viruses to elude the
immune system of the host by surrounding
itself with the host envelope
 Enveloped viruses may be more vulnerable
to chemical agents(chlorine, hydrogen peroxide, and phenol)
 They do not survive well on surfaces
Viral Life Cycle – Factors
Influencing the Life Cycle
 Nucleic acid
 Enveloped or naked
 Shape
 Host
Host Range
 Primates
 Vertebrates ( birds)
 Plants
 Insects
 Bacteria
Bacteriophages
 Viruses that infect bacterial cells
 Genome can be DNA or RNA
 Bind to specific receptors that are
proteins or carbohydrates in the bacterial
cell wall
Bacteriophage structure
Bacteriophages
PhiX174 – Spherical
Bacteriophage
 Interesting to study because
of its overlapping genes which
is a model of efficiency
T- 4 Bacteriophage
 Studied by Luria and Delbruck at Cold
Spring Harbor
 Ds DNA virus
 168, 800 base pairs
 Phage life cycles studied by Luria and
Delbruck
Filamentous phages
Fd
Filamentous
Circular ss DNA
Lies in the middle of
the filament
 Infects through the
pilus
 Create a symbiotic
relationship with the
host
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M 13
 Used for Genetic
engineering
experiments
Bacteriophages Life Cycles
 Lytic – They attach and enter the bacterial
cell. Complete their life cycle by bursting
the bacterial cell – “ lysis”
 Lysogenic – They attach and enter the
bacterial cell. The virus then integrates
into the bacterial cell as a “prophage”
Process of Infection
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Attachment
Injection
Hostile take over( lytic)
Integration( lysogenic) – Genetic control
Early genes/late genes
Replication of nucleic acid
Production of viral proteins
Assembly
Lysis
Lytic Life Cycle( Virulent)
 The viral genome contains a promoter that
attaches to host cell RNA Polymerase
 Early genes code for those proteins that shut
down the host, replicate nucleic acid, and code for
vital proteins
 Nuclease genes, are capable of digesting host
DNA so that the bases can be used for the
production of new virions
 Late genes code for viral capsid and for those
proteins that lead to lysis of the host cell
Lytic Cycle
 Strict control
 Do not want to lyse the cell prior to the
completion of assembly
 Usually only one virus in a cell at a time
 Recombination occurs between the two
viruses if more than one is present
Lysogenic Life Cycle
 Bacteriophages that do not lyse the bacteria cell
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are referred to as temperate
Lysogenic bacteria contain a copy of the virus
which is non infective and is known as the
prophage
The prophage can remain inactive through many
cell division
Bacteria can switch between lytic and lysogenic
life styles
When the host is stressed or damaged by
mutagens, this stimulates the prophase to excise
itself and
Generalized Transduction
 Any part of bacterial genome can be
transferred
 Occurs during lytic cycle
 During viral assembly, fragments of host
DNA mistakenly packaged into phage head
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generalized transducing particle
Generalized transduction
Specialized transduction
Lambda Phage
 Bacteriophage Lambda is a temperate
phage meaning that it can undergo either a
lytic or lysogenic cycle
 The phage regulates this cycle genetically
through a switch
Attachment
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Bacteriophage Lambda binds to the target E. coli
cell, the tail tip binding to a maltose receptor.
The linear phage genome is injected into the cell,
and immediately circularises.
Transcription starts
There are two regulatory viral proteins, CI and
Cro
CI and Cro compete for the operator promoter
sites on the phage DNA
When the bacterial host is healthy CI
accumulates and the lambda integrates into the
bacterial genome and stays in this position
Control of lysogeny and lytic
cycle
 Genes needed to
establish lysogeny
 cI yes
 cII yes
 cIII yes
 Genes needed for
maintenance of lysogeny
 cI yes
 cII no
 cIII no
Insertion sequences
Genetic Control of Lytic and
Lysogenic Phage
Lytic vs. Lysogenic
 When Cro is low, CI maintains the lysogenic
life style
 When Cro accumulates due to damage of
the host DNA or other unsuitable
environmental conditions, the virus
switches to the lytic life style
 This activates promoters for phage
replication and protein synthesis
 Serves as a model for understanding viral
infectivity and genetic control
Integration
 Integration of bacteriophage lambda requires one
phage-encoded protein - Int, which is the
integrase - and one bacterial protein - IHF, which
is Integration Host Factor.
 Both of these proteins bind to sites on the P and
P' arms of attP to form a complex in which the
central conserved 15 bp elements of attP and attB
are properly aligned.
 The integrase enzyme carries out all of the steps
of the recombination reaction, which includes a
short 7 bp branch migration.
Enzymes and Recombination
 The strand exchange reaction involves
staggered cuts that are 6 to 8 bp apart
within the recognition sequence.
 All of the strand cleavage and re-joining
reactions proceed through a series of
transesterification reactions like those
mediated by type I topoisomerases.
Excision of bacteriophages
 Excision of bacteriophage lambda requires two phage-
encoded proteins:
 Int (again!) and Xis, which is an excisionase. It also requires
several bacterial proteins.
 In addition to IHF, a protein called Fis is required.
 All of these proteins bind to sites on the P and P' arms of
attL and attR forming a complex in which the central
conserved 15 bp elements of attL and attR are properly
aligned to promote excision of the prophage.
Lytic Cycle
 Lytic Lifestyle
 The 'late early' transcripts -genes for replication
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of the lambda genome.
The lambda genome is replicated in preparation
for daughter phage production.
Transcription from the R' promoter can now
extend to produce mRNA for the lysis and the
structural proteins.
Structural proteins and phage genomes self
assemble into new phage particles.
Lytic proteins build sufficiently far in
concentration to cause cell lysis, and the mature
phage particles escape.
Bacteriophage growth curve
Plaque Assay
 Eclipse Period- Penetration through
biosynthesis
 Latent-Spans from penetration up to the
point of phage release
Lambda and Plaques
 The plaque produced by Lambda had a
different appearance on the Petri Dish.
 It is considered to be turbid rather than
clear
 The turbidiy is the result of the growth of
phage immune lysogens in the plaque
 The agar surface contains a ratio of about
a phage /107 bacteria
Plaque assay
Methodology
 Grow bacteria to log phase
 Prepare dilutions of bacteriophage
 Mix bacteria with viruses or overlay
bacteria with suspension of viral particles
 Incubate
 Count infectious particles based upon the
number of plaques
 Plaques are clear areas indicating lysis of
infected cells
Plaque Assay
MOI
 Average number of phages /bacterium
 After several lytic cycles the MOI(
multiplicity of infection) gets higher due to
the release of phage particles
Horizontal Transfer of Genes
 The transfer of genes or blocks of genes
( pathogenicity islands) from bacterium to
bacterium or virus to bacteria or virus to
virus
 Has resulted in many changes in microbes
that have led to increase in pathogenicity
and accumulation of virulence genes, not
just resistance
Streptococcus pyogenes
 There are 15 prophages that have been
identified in E. coli
 These prophages belong to the group
Siphoridae
 All but one of these produce a toxin
 In both strep and staph – the prophage is
found at the site of recombination
Virulence and Streptococcus
pyogenes
 Streptococcal pyrogenic exotoxins(SPE)
contribute to the diverse symptoms of a
streptococcal infection.
 These antigens compare to Staphylococcal
antigens of the same type.
 The A + C genes coding for these toxins were
horizontally transferred from strain to strain by a
lysogenic bacteriophage.
 In addition the genes contributed by the phages
produce hyaluronidase, mitogenic factor, and
leukocyte( WBC) toxins
Pathogens with bacteriophages
that cause complications
 Corynebacterium diphtheriae
 Vibrio cholerae
 enterotoxogenic E. coli
 Staphylococcus aureus
 Clostridium botulinum
 Staphylococcus aureus and Streptococcus
pyogenes: Toxic shock syndrome
Toxic shock
Parvoviridae
 Viral family: Parvoviridae
 single-stranded DNA; naked; polyhedral
capsid
 Size: 18-25nm
 Examples and diseases: parvoviruses
(roseola, fetal death, gastroenteritis; some
depend on coinfection with adenoviruses)
Papovaviridae
 double-stranded, DNA; naked;
polyhedral capsid
 Viral family: Papovaviridae; circular dsDNA
 Size: 40-57nm
 Examples and diseases: human papilloma
viruses (HPV; benign warts and genital
warts; genital and rectal cancers)
Adenovirus
 Viral family: Adenoviridae; dsDNA
 Size: 70-90nm
 Examples and diseases: adenoviruses (see
Fig. 1E) (respiratory infections,
gastroenteritis, infectious pinkeye, rashes,
meningoencephalitis)
Pox Viruses
 double-stranded, circular DNA; enveloped;
complex
 Viral family: Poxviridae
 Size: 200-350nm
 Examples and diseases: smallpox virus (smallpox),
vaccinia virus (cowpox), molluscipox virus
(molluscum contagiosum-wartlike skin lesions
Herpes and Hepadnaviridae
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double-stranded DNA; enveloped; polyhedral capsid
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Viral family: Herpesviridae
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Size: 150-200nm
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Examples and diseases: herpes simplex 1 virus (HSV-1; most oral herpes;
see Fig. 1H), herpes simplex 2 virus (HSV-2; most genital herpes), herpes
simplex 6 virus (HSV-6; roseola), varicella-zoster virus (VZV; chickenpox
and shingles), Epstein-Barr virus (EBV; infectious mononucleosis and
lymphomas), cytomegalovirus (CMV; birth defects and infections of a
variety of body systems in immunosuppressed individuals)
Viral family: Hepadnaviridae
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Size: 42nm
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Examples and diseases: hepatitis B virus (HBV; hepatitis B and liver cancer)
Picornaviruses
 (+)single-stranded RNA; naked; polyhedral
capsid
 Viral family: picornaviridae
 Size: 28-30nm
 Examples and diseases: enteroviruses
(poliomyelitis), rhinoviruses (most frequent cause
of the common cold), Norwalk virus
(gastroenteritis), echoviruses (meningitis),
hepatitis A virus (HAV; hepatitis A)
Togaviruses
 (+)single-stranded RNA; enveloped; usually a
polyhedral capsid
 Viral family: Togaviridae
 Size: 60-70nm
 Examples and diseases: arboviruses (eastern
equine encephalitis, western equine encephalitis),
rubella virus (German measles)
- Strand viruses
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(-) strand; multiple strands of RNA; enveloped
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Viral family: Orthomyxoviridae
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Size: 80-200nm
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Examples and diseases: influenza viruses A, B, and C (influenza)
Viral family: Bunyaviridae
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Size: 90-120nm
Examples and diseases: California encephalitis virus (encephalitis);
hantaviruses (Hantavirus pulmonary syndrome, Korean hemorrhagic fever;
see Fig. 1G)
Viral family: Arenaviridae
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Size: 50-300nm
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Examples and diseases: arenaviruses (lymphocytic choriomeningitis,
hemorrhagic fevers)
HIV
 produce DNA from (+) single-stranded RNA
using reverse transcriptase; enveloped; bulletshaped or polyhedral capsid
 Viral family: Retroviridae
 Size: 100-120nm
 Examples and diseases: HIV-1 and HIV-2 (HIV
infection/AIDS; see Fig. 3A); HTLV-1 and HTLV-2
(T-cell leukemia)
Key steps in viral infections
 Adsorption
 Fusion/Uncoating
 Hostile Takeover
 Early Genes for Replication of Virus
 Late Genes for Proteins and Capsid
 Lysis
Animations of adsorption
 Flash animation showing adsorption of an
enveloped virus.
 Flash animation showing adsorption of a
naked virus.
 QuickTime movie showing adsorption of an
enveloped virus.
 QuickTime movie showing adsorption of a
naked virus.
Fusion and uncoating
 Flash animation showing penetration and uncoating
of an enveloped virus entering by fusion.
 Flash animation showing penetration and uncoating
of an enveloped virus entering by endocytosis.
 QuickTime movie showing penetration and
uncoating of an enveloped virus entering by fusion.
 QuickTime movie showing penetration and
uncoating of an enveloped virus entering by
endocytosis
Animations of Replication
 Flash animation showing replication of an
enveloped virus.
 Flash animation showing replication
of a naked virus.
 QuickTime movie showing replication of an
enveloped virus.
 QuickTime movie showing replication
of a naked virus.
Release of Virus
 Flash animation showing release of a naked
virus by cell disintegration.
 QuickTime movie showing release of a
naked virus by cell
RNA Virus
Global Pandemic
Influenza
 Orthomyxovirus –
medium sized
enveloped(-) sense
RNA – leads to
recombination
 They have a
segmented genome
 Broad host range
 Whales, swine,birds,
and humans
Antigens
 Influenza neuraminidase
exists as a mushroomshape projection on the
surface of the influenza
virus.
 It has a head consisting of
four co-planar and roughly
spherical subunits, and a
hydrophobic region that is
embedded within the
interior of the virus'
membrane.
 It is comprised of a single
polypeptide chain
Hemagglutinin
 HA binds to an as yet unidentified
glycoprotein which is present on the
surface of its target cells.
 This causes the viral particles to stick to
the cell's surface. The cell membrane then
engulfs the virus and the portion of the
membrane that encloses
 It pinches off to form a new membranebound compartment within the cell called
an endosome, which contains the engulfed
virus.
Avian flu
 Avian influenza is an
infection caused by avian
(bird) influenza (flu)
viruses.
 These influenza viruses
occur naturally among
birds. Wild birds worldwide
carry the viruses in their
intestines, but usually do
not get sick from them.
 However, avian influenza is
very contagious among
birds and can make some
domesticated birds,
including chickens, ducks,
and turkeys, very sick and
kill them.
Infection
 Infected birds shed influenza virus in their saliva,
nasal secretions, and feces.
 Susceptible birds become infected when they have
contact with contaminated secretions or
excretions or with surfaces that are contaminated
with secretions or excretions from infected birds.
 Domesticated birds may become infected with
avian influenza virus through direct contact with
infected waterfowl or other infected poultry, or
through contact with surfaces (such as dirt or
cages) or materials (such as water or feed) that
have been contaminated with the virus.
Infection with avian influenza viruses
Strains of Avian Flu
Hepatitis B Virus
 Hepatitis B is a DNA
Virus of the
Hepadnaviridae family
of viruses.
 It replicates within
infected liver cells
(hepatocytes ). The
infectious ("Dane")
particle consists of an
inner core plus an
outer surface coat.
Hepatitis B antigens
 When the virus enters the body of a new host it's
initial response, if it's gets past the immune
system, is to infect a liver cell. To do this the
virus attaches to a liver cells membrane and the
core particle enters the liver cell. The core
particle then releases it's contents of DNA and
DNA polymerase into the liver cell nucleus.
 From within the cell nucleus the hepatitis B DNA
causes the liver cell to produce, via messenger
RNA; surface (HBs) proteins, the core (HBc)
protein, DNA polymerase, the HBe protein, HBx
protein and possibly other as yet undetected
proteins and enzymes.
HbS Ag
 Hepatitis B Surface protein(s). (HBsAg)
The outer surface coat composed of hepatitis B
surface proteins is produced in larger quantities
than required for the virus to reproduce. The
excess surface proteins clump together into
spherical particles of between 17-25nm in
diameter but also form rods of variable length. In
some cases these particles encapsulate a core
particle and produce a complete, and infectious,
virus particle that enters the blood stream and
can infect other liver cells. The excess spheres,
rods and also complete viral particles enter the
blood stream in large numbers and are easily
detectable. It does however take a while for
these proteins to appear.
Viral Structure
Genes and Gene Products
Viral Replication
HBV
Picornaviruses
Polio virus
 Picornaviruses are non-
enveloped.
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As a consequence,
they are resistant to
lipid solvents like
ether and chloroform
which would destroy
an enveloped virus.
 Picornaviruses have an
icosahedral capsid.
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Poliovirus
 An enterovirus
 Enters the body through inhalation of
water
 Used to be from swimming pools
 Causes encephalitis
 CNS involvement from mild to severe.
 Presents with fever, malaise,cnills, and
neruological effects
Rhinoviruses
 Picornaviruses replicate in
the cell cytoplasm.
 Picornaviruses can
replicate in an
enucleated cell (a cell
that has had its nucleus
removed) because they
only need host cell
machinery and
components that are
present in the
cytoplasm. They don’t
require anything found
in the host cell nucleus
Echoviruses
 Picornaviruses have a
positive sense RNA
genome.
 The picornavirus
genome has the same
sense (polarity) as
mRNA. The genome
alone can use the host
cell's machinery to
make whatever is
needed to replicate the
virus. If only genomic
RNA is injected into a
host cell, it is infectious
and the cell produces
progeny virions.
Rhabdovirus - Rabies
Rabies
 Ss RNA virus
 Secreted in the saliva of
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infected animals
Transferred by bite or
scratch
75,000 cases if rabies
occur around theworld in
humans
Virus travels on axons of
motor or sensory neurons
Spreads to brain with
serious consequences
National Rabies Management
Program
 The development of a natural vaccine
 Research completed at Wistar Institute in
Philadelphia, Pa.
 Vaccine is an oral type placed in natural
habitats
 Food or bait is desirable to many animals
such as fox, bear, otter, beaver, and
racooons
Distribution of Rabies in the
United States